The occlusion of blood vessels and capillaries by blood clots is the most frequent cause of mortality in the developed world. The process of blood clotting, called thrombosis, involves a complex system of interacting enzyme factors, each of which is converted by other enzymes from an inactive to an active form. As a result of the overall activity of this system, protein fibers called fibrin become enmeshed in a mass which curtails blood flow at the point of the thrombosis. Where such thrombosis occurs at the site of a cut, the effect is the protective reduction of blood loss through bleeding. But where such thrombosis occurs in a uncontrolled manner in major arteries supplying the lungs, brain or other vital organs, the result may be paralysis, loss of neural function, or death, unless the fibrin clot can be removed expeditiously.
The removal of fibrin clots may sometimes be effected by surgical means, but this is not always practicable. Fortunately, however, there is a physiological system that can reverse the thrombosis process by dissolving the fibrin clots. (For a review of this system, see Gronow and Bliem, 1983, Trends in Biotech. 1: 26-29) At the heart of this system is plasmin, an enzyme that can degrade the large, insoluble fibrin mass to small, soluble components. Plasmin, however, is normally present in the body in an inactive precursor form, called plasminogen. Plasminogen may be converted to plasmin through a single enzymatic cleavage of its polypeptide structure, by a member of a family of specific hydrolase enzymes called plasminogen activators (PAs). These PAs are believed to be serine hydrolases, because they are inhibited by the relatively serine-specific enzyme inhibitor diisopropyl fluorophosphate (DFP).
On the basis of neutralization by specific antisera and affinity for fibrin, PAs may be divided into two broad categories. The first group is related to urokinase, which can be isolated from human urine and is produced by the cells which line the kidney tubules. These "urokinase-like" PAs (UPAs) may be readily identified by the fact that their activity is neutralized by anti-urokinase antiserum. Furthermore, UPAs, another example of which is streptokinase of bacterial origin, can be distinguished from the other broad category of PAs by the fact that they do not bind to fibrin. This observation is of practical importance in the therapeutic removal of fibrin clots; since urokinase and streptokinase have no affinity for fibrin, much larger quantities of these UPAs must be used to dissolve fibrin clots. Furthermore, the large excess of UPAs that is required may produce undesired systemic plasminogenolysis, with concomitant internal hemorrhaging.
The second broad category of PAs is the group of "tissue-like" PAs (TPAs). This type of PA, which is produced by human lung and many other tissues and by numerous mammalian tumor cells, is not neutralized by anti-urokinase antiserum. Antiserum prepared against one TPA will neutralize other TPAs as well, but not the UPAs. The TPA class of plasminogen activators also binds to fibrin clots. As a result, the conversion of plasminogen to plasmin by TPAs tends to occur only at the site where it is needed, rather then systemically. Therefore, TPAs may be used in much lower therapeutic concentrations, a fact which would make thrombosis therapy with TPAs much less costly and far safer.
Plasminogen activators may be isolated from numerous sources. They may be found, for example, in blood, tears, saliva, urine, semen and in cerebrospinal or other body fluids (Gronow and Bliem, 1983, Trends in Biotech. 1: 26-29) They may also be found in many tissues and cells. Wilson et al. (Cancer Research, 1980, 40: 933-938) have found PAs in human embryo fibroblasts, bladder, lung, brain, thyroid, kidney, skin, foreskin fibroblasts and epithelium, and fallopian tube epithelium.
It has long been known that many tumor cells also produce PAs (Rifkin et al., 1974, J. Exp. Med. 139: 1317-1328). Many investigators believe that the ability of tumor cells to produce PAs contributes to their tendency to metastasize and thus, at least in part, facilitates the spread of neoplastic disease. In any event, the fact that tumor cells are easily cultured and that they often produce PAs has made possible the production of substantial quantities of PAs from tissue culture systems.
Dano and Reich (1978, J. Exp. Med. 147: 745-757) have described the production of PAs by cultured C57 black mouse embryo cells that were transformed by infection with murine sarcoma virus. In subsequent studies, Dano et al. (1980, Biochim. Biophys. Acta 613: 542-555) purified PA to homogeneity from cultures of mouse fibroblasts transformed with murine sarcoma virus and showed that its subunits corresponded in electrophoretic mobility in sodium dodecyl sulfate to those of human urokinase.
The recovery of TPA from medium harvested from a human melanoma cell line culture (called the Bowes melanoma cell line) has been reported by Rijken and Collen (1981, Progr. Chem. Fibrinolysis Thrombolysis 5: 236-239). This TPA, which was purified by a variety of chromatographic techniques resulting in a recovery of 1 to 4 mg protein per 10 liters of medium and a 260-fold increase in the specific activity of the purified product as compared to the specific activity in the initial growth medium, was immunologically unrelated to urokinase and absorbed almost completely to fibrin. More recently, Pennica, et al. (1983, Nature 301: 214-221) have cloned the TPA genome from the Bowes melanoma cell line into an E. coli bacterium and have achieved the expression of TPA.
Both a TPA and a UPA have been recovered from supernatants of confluent cultures of a rat prostate adenocarcinoma cell line, PA III (Strickland et al., 1983, Biochemistry 22: 4444-4449). The UPA (45,000 daltons, molecular weight) was purified chromatographically resulting in a recovery of 10 .mu.g protein per 300 ml medium (at a 66% yield) and a 364-fold increase in specific activity. A second protein (80,000 daltons, molecular weight), believed to be TPA, was not extensively investigated.
Although cultured cells, such as the Bowes melanoma cell line, may produce substantial quantities of TPA, efforts have been made to increase the TPA output of tissue culture systems. In one such effort, Hull et al. (U.S. Pat. No. 3,904,480) developed a method whereby the PA production of cultured kidney cells from a variety of animal species could be increased by maintaining the cell in medium containing low levels of mitotic inhibitors, such as colchicine or vinblastine. No mention was made about whether TPA or UPA was obtained, although since the cells were from kidney, it was probably the latter enzyme. The toxic effects of the sustained exposure to the mitotic inhibitors on the ultimate longevity of the cells was not examined. The degree of enhancement of PA production by their method, compared to production by untreated cells, was also not stated.
In U.S. Pat. No. 4,232,124, Mann describes substantially elevated production of TPA by cultured human diploid fibroblast cells of the MCR-5 strain, in the presence of a TPA inducer. Using lactalbumin hydrolysate as an inducer of TPA synthesis, Mann obtained a dose-dependent response. In the presence of lactalbumin hydrolysate, however, cultures degenerated and precipitated in 6 to 12 days. In contrast, cultures not treated with the inducer could be harvested with repeated medium changes for over a month.
Horiguchi et al. (U.S. Pat. No. 4,328,314) have developed a method for obtaining a 30% to 180% increase in PA production by cultured renal cells of monkey or human origin. This enhanced production of what was most probably UPA was obtained by placing low levels of organic acids, such as fumaric, malic or succinic acid, in the culture medium. Any possible adverse effects on the longevity of treated cells were not described.
From what can be determined based upon the tissue culture references, supra, the harvesting of PA-containing medium from cell cultures (i.e., conditioned medium) can be continued only for a period of about two to perhaps six weeks. After a period of growth which is determined by the characteristics of the cells and the culture medium and conditions, cell degeneration and detachment occurs. When that point is reached, PA production ceases, and the culture vessels must be cleaned, sterilized and reseeded with fresh cells. This relatively limited lifespan of most culture systems is an annoying and costly impediment to efficient TPA production.